Hi

What you are suggesting is theoretically valid to detect and capture the primary Ab but it would not prevent its binding to non specific structures on your tissue specimens as this step always comes after the incubation of the primary with the tissue for some time (up to overnight incubation in 4 C).

The serum will block the primary antibody from binding to any other antigenic structures except for your antigen of interest and hence it is mostly recommended to apply normal serum from the species in which the primary Ab was made in. Therefore it is recommended to incubate the primary Ab along with the normal serum at the same step.

Regards,

Bassem

Hello

i agree with Bassem Refaat , Usually, 5- 10% normal serum is used. It should come from the same host species as the secondary antibody. The idea is that the IgG in serum occupies “sticky” binding sites to prevent non-specific binding. It therefore shouldn’t come from the same host species as the primary antibody. I support what she said , should be incubate the primary Ab along with the normal serum at the same step.

Good Luck

If you're worried block with non-immune serum from the primary and secondary species.

Thanks to all, for your answers, I see that the serum made in the host where the secondary AB is produced will bind to sticky sites that could potentially be occupied by the primary antibody. That make sense. I suppose that non immune serum is serum without the Ig fraction? it seems a nice suggestion from Neil, i will try in a reaction against GABA in which I have a low signal/noise ratio. Astrid !!! nice to see you here, how are you doing? I suppose that for the majority of reactions it will be fine with SFB or BSA.